Humán és bakteriális hősokkfehérjék komplementaktiváló képességének összehasonlító vizsgálata = Study on the complement activating ability of human and bacterial heat-shock proteins

A kutatási periódusban 4 célkitűzésben vizsgáltuk humán és bakteriális hősokkfehérjék komplementaktiváló képességét. A komplementaktiváció legfontosabb regulátorainak, a Hsp60/65 ellens antitestek vonatkozásában azt kaptuk, hogy jelentős reguláló tényező az IL-6 promóter -174-es polimorfizmusa. Kimutattuk továbbá, hogy az anti-Hsp60 autoantitestek a természetes autoantitest repertoárba tartoznak, valamint epitóp szinten is elkülöníthetők a az anti-Hsp65 antitestektől. Eredményeink jelentőségét az adja, hogy eddig csak korlátozott ismeretanyaggal rendelkeztünk az anti-Hsp autoantitestek összefüggéseire és regulációjára vonatkozóan. A pályázat támogatásával az erre vonatkozó részletes immunológiai ismeretanyag gazdagodott. A Hsp70 komplementaktiváló képességének in vitro vizsgálatát megnehezítette a rekombináns fehérjék endotoxin szennyezettsége, ami mellett nem tudtuk megítélni a komplementaktiváció pontos mechanizmusát. A Hsp70 további vizsgálatát in vivo, klinikai beteganyagon valósítottuk meg, ezek a megfigyeléseink lehetővé teszik egy későbbi, in vivo komplementaktivációra vonatkozó munkahipotézis kialakítását. Eredményeinknek gyakorlati vonatkozását az adja, hogy a hősokkfehérjék immunológiai szerepe egyre több klinikai állapotban nyer bizonyítást, és az ezzel kapcsolatos részletes szabályozó folyamatok jellemzése hozzájárul az adott kórállapotok pontosabb megértéséhez. | The complement (C) activating ability of human and bacterial heat shock proteins was investigated along four objectives in this project. Investigating the main regulator of Hsp60/Hsp65 induced C activation, i.e. anti-Hsp antibodies, the primary regualting role of the IL-6 -174 promoter polymorphism was shown. Furthermore, evidences were obtained about the natural autoantibody properties of anti-Hsp60 IgM antibodies. These antibodies can also be distinguished from anti-Hsp65 antibodies based on their epitope specificities. There were only limited data available until now on the regulation and associations of anti-Hsp autoantibodies. With the support of the current research grant detailed immunological knowledge has been obtained on the above topic. The in vitro investigations on the Hsp70 induced C activation was hampered by the endotoxin contamination of the recombinant Hsp70 preparations. Therefore, our subsequent in vivo investigation on Hsp70 was done in clinical studies. The results of these provide base for follow-up in vivo research to determine the exact mechanism of Hsp70 induced C acitvaiton in vivo. The physiological relvance of our results is related to the fact of recent observations about the roles of heat shock proteins in different human diseases. Understanding the precise immunological mechanisms behind these associations will help in the future to appreciate the clinical consequences of Hsps in more depth

FcRn Overexpression in Transgenic Mice Results in Augmented APC Activity and Robust Immune Response with Increased Diversity of Induced Antibodies

Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance

Characterization of the Rabbit Neonatal Fc Receptor (FcRn) and Analyzing the Immunophenotype of the Transgenic Rabbits That Overexpresses FcRn

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits – having one extra copy of the FcRn when hemizygous and two extra copies when homozygous - showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies

Transgenic rabbits that overexpress the neonatal Fc receptor (FcRn) generate higher quantities and improved qualities of anti-thymocyte globulin (ATG).

Immune suppression with rabbit anti-thymocyte globulin (rATG) is a well-established therapeutic concept for preventing host rejection of transplanted organs and graft versus host disease. Increasing the efficiency of rATG production by reducing the number of animals would be highly beneficial to lower cost and to improve quality standards. We have developed transgenic (Tg) mice and rabbits that overexpress the neonatal Fc receptor (FcRn) and have shown an augmented humoral immune response in these animals. To test whether our FcRn Tg rabbits produced rATG more efficiently, we immunized them and their New Zealand White controls with live Jurkat cells. By day 21 after immunization, Tg animals produced significantly, 1.5 times higher amount of total IgG compared to their wt littermates. Also, the binding efficiency of Tg sera to Jurkat cells and their complement-mediated cytotoxicity was significantly higher. The purified Tg IgG preparation contained 2.6 the amount of Jurkat specific IgG as the wt preparation analyzed by complement-mediated lysis, suggesting greater antigen-specific B cell activation in the Tg rabbits. To test this hypothesis, immunization with ovalbumin and human α1-antitrypsin was performed, resulting in significantly greater numbers of antigen-specific B-cells in the FcRn Tg rabbits as compared with wt controls. The shift towards significantly larger populations of antigen-specific B cells relative to the non-specific B cell pool is further corroborated by our previous findings in FcRn Tg mice. Consequently, our FcRn Tg rabbits have the potential to offer substantial qualitative and quantitative improvements for the production of rATG and other polyclonal or monoclonal antibodies

Complement activated cytotoxicity of rATG from Tg and wt rabbits.

<p><b>A.</b> Complement activated cytotoxicity of pooled immune serum samples collected from wt and Tg rabbits on day 21 of immunization. Tg-pool of immune sera was more efficient in killing Jurkat cells at all dilutions. <b>B.</b> Concentration dependent induction of complement activated cytotoxicity by protein-G purified IgG collected on day 21 of immunization from wt and Tg rabbits (ATG-Fresenius S IgG was used as reference). We observed more efficient cytotoxicity of the Tg samples at all concentrations, and when cytotoxicity curves were analyzed with non-linear regression analysis, data showed that 20% cytotoxic activity was achieved by adding 259 µg/ml, 273 µg/ml or 99 µg/ml IgG preparations from the wt IgG, ATG-Fresenius or Tg IgG preparations, respectively, indicating that Tg IgG mediated cytotoxicity was more than 260% more efficient than its wt control. Percent of lysed cells was determined by flow cytometry analyzing propidium iodide incorporation (samples were analyzed in triplicate and error bars indicate standard deviations of these measurements).</p

Kinetics of Jurkat specific immune response in Tg and wt rabbits.

<p><b>A.</b> Total IgG concentrations of serum samples collected from rabbit FcRn transgenic (Tg) and wild type (wt) rabbits immunized with Jurkat T cells (determined by ELISA assay). <b>B.</b> Development of immune response to Jurkat T cell surface antigens during immunization (determined by flow cytometry binding assay of 1∶250-fold diluted individual rabbit serum samples). Each dot represents one animal as an average of three measurements; (* <i>P</i><0.05; ** <i>P</i><0.01).</p

FcRn Tg rabbits have augmented antigen specific B-cell activation.

<p>Immunization with ovalbumin (OVA) and human α1-anti-trypsin (hA1AT) resulted in generally higher level IgM and IgG titers measured by ELISA, more antigen specific IgM and IgG producing B cells determined by ELISPOT, and larger spleen in Tg rabbits as compared to their wt controls (* <i>P</i><0.05; ** P<0.01).</p

Binding of Protein-G purified, pooled Tg and wt IgG antibodies to Jurkat cells.

<p>The binding capacity of the Tg IgG analyzed by flow cytometry in a concentration range from 10 - 0.039 µg/ml was higher at all concentrations compared to their wt controls. The non-linear regression analysis indicated that binding activities of MFI 20 was achieved at concentration of 6.92 µg/ml or 4.23 µg/ml, in cases of wt or Tg preparations, respectively, indicating that Tg IgG binding was more than 60% higher than its wt control.</p

Binding of rATG to Jurkat cells from serum samples at different dilutions.

<p>Flow cytometry binding assay was performed and the geometric means of fluorescence intensities obtained are presented for different immune sera dilutions. Antigen binding of individual samples at immune serum dilutions of 1∶500 (<b>A</b>), 1∶1000 (<b>B</b>), 1∶5000 (<b>C</b>) and 1∶10000 (<b>D</b>) are shown in small inserts. Data show that Tg sera bindings were significantly higher than its wt controls in all dilutions. Each dot represents one animal as an average of three measurements (* <i>P</i><0.05; *** <i>P</i><0.001).</p

Serum soluble E-selectin and NT-proBNP levels additively predict mortality in diabetic patients with chronic heart failure.

This study demonstrated a weak correlation of sE-selectin level with inflammatory markers and prediction of short-term mortality in diabetic CHF patients. Elevated serum sE-selectin levels and concomitantly increased NT-proBNP concentrations have additive predictive power in CHF. This suggests that parallel activation of various pathophysiological pathways confers increased risk of adverse outcome in CHF